Cell surface glycoproteins are believed to play a role in cell recognition events during both normal and abnormal growth and differentiation. A key step in the assembly of N-linked glycoproteins is the co-translational transfer of a preassembled oligosaccharide chain from dolichylpyrophosphate to an appropriate -Asn-X-Ser/Thr- site in the growing polypeptide chain. If the mature N-linked glycoprotein is destined to be of the polymannose type, subsequent processing in the Golgi complex is very limited. In contrast, if it is destined to be of the complex type extensive processing, involving removal of all but three of the Man residues and the addition of "capping sugars", occurs. The proposed studies will focus on three events in this overall process. First, the enzyme that catalyzes the transfer of the oligosaccharide chain, oligosaccharyltransferase, will be isolated from hen oviduct and characterized. Antibody to the enzyme and active site photoreactive peptide substrates will be employed in the isolation. The antibody will also be used to study the biosynthesis of this enzyme in oviduct cells. Peptides that inhibit the enzyme in vitro will be developed and studied as in vivo inhibitors of glycosylation in a number of cell types. Second, efforts will be made to better understand why certain -Asn-X-Ser/Thr- sites in proteins are not acted upon by oligosaccharyltransferase in vivo. Using ovalbumin as a model it will be determined if spatial factors prevent glycosylation of a second acceptor site when ovalbumin is synthesized in vivo. In addition, the role of temporal factors in the glycosylation of the second site of ovalbumin synthesized in vitro will be investigated. Third, the factors that control the processing of oligosaccharide chains after their transfer to protein will be studied. It is known that the polymannose chain of native bovine pancreatic RNase B is not processed by Golgi membranes from bovine pancreas, but is processed by rat liver membranes. The possibility will be investigated that this is due to differences in the properties of the GlcNAc transferase I from these 2 sources. In addition, efforts will be made to identify structural features in the polypeptide chain of bovine pancreatic RNase B that prevent its oligosaccharide chain from being processed by the pancreatic enzyme. Finally, these processing studies will be extended to 3 other high mannose glycoproteins and Golgi membranes from sources that are either homologous or heterologous with respect to the source of glycoproteins.